CN112334129A

CN112334129A - Composition comprising amino acids for preventing and treating liver diseases

Info

Publication number: CN112334129A
Application number: CN201980042577.XA
Authority: CN
Inventors: 保罗·卢卡·马里亚·焦尔杰蒂
Original assignee: Professional Nutrition International Co ltd
Current assignee: Professional Nutrition International Co ltd
Priority date: 2018-06-27
Filing date: 2019-04-02
Publication date: 2021-02-05
Also published as: IT201800006725A1; CA3104839A1; BR112020026431A2; JP7347744B2; AU2019293293A1; RS61613B1; ES2858355T3; MX2020013471A; PH12020552254A1; ZA202007834B; LT3586837T; US20210260011A1; EP3586837A1; EP3586837B1; EA202092749A1; JP2021529731A; DK3586837T3; CL2020003209A1; KR20210025611A; WO2020003013A1

Abstract

A composition for use in the prevention and/or treatment of liver disease in a mammal, the composition comprising an active agent comprising the amino acids leucine, isoleucine, valine, threonine, lysine and the carboxylic acids citric acid, succinic acid, malic acid.

Description

Composition comprising amino acids for preventing and treating liver diseases

Technical Field

The present specification generally relates to compositions comprising amino acids. More specifically, the present specification relates to compositions comprising amino acids for the prevention and/or treatment of liver diseases.

Background

Liver diseases are among the most frequently occurring diseases caused by a variety of adverse environmental conditions, such as parasites and viruses, drugs, toxic substances, alcoholism and smoking. It is often a chronic disease that presents with worsening clinical features. Chronic liver disease is characterized by: liver parenchyma is gradually destroyed over time, has an inflammatory response and fat accumulation, has fibrotic lesions and activation of cellular transformation, leading to carcinogenesis in many cases. Thus, liver disease may include steatosis, fibrosis, cirrhosis and hepatocellular carcinoma as progressive clinical disorders of the liver. Cirrhosis is the result of acute and chronic liver disease and is characterized by: liver tissue is replaced by fibrotic scar tissue and regenerative nodules, resulting in progressive loss of liver function. Fibrosis and nodular regeneration result in loss of the normal microscopic lobular structure of the liver. Fibrosis represents the growth of scar tissue resulting from, for example, infection, inflammation, injury, and even healing. Over time, fibrotic scar tissue slowly replaces normal functional liver tissue, resulting in a decrease in the amount of blood flowing to the liver, making the liver unable to adequately treat nutrients, hormones, drugs and poisons present in the bloodstream. The more common causes of cirrhosis include alcoholism, hepatitis c virus infection, ingestion of toxins, and there are many other possible causes. As mentioned below, the most epidemiologically relevant disorder that progresses to cirrhosis and possibly to cancer is the increase in body fat and the associated accumulation of liver fat. This fat accumulation can be attributed to two major clinical conditions: alcohol abuse and obesity.

Excessive and prolonged drinking can lead to Alcoholic Liver Disease (ALD), a major global health problem. The pathological features of ALD develop over a long period of time, including hepatic steatosis, steatohepatitis, cirrhosis, and through to cancer.

Ethanol pathogenesis is caused by a variety of factors. Early events such as mitochondrial damage, Reactive Oxygen Species (ROS) generation and fat accumulation appear to be a direct consequence of ethanol metabolism and are a common feature between ALD and nonalcoholic liver disease or NAFLD (see below) (Mantena et al 2008).

The cellular defense mechanisms against the deleterious effects of alcohol are not well understood. Autophagy, an important degradation cellular pathway to digest cellular proteins and organelles to gain energy or to eliminate damaged cellular structures, is thought to play a role in ALD, but the understanding of these mechanisms remains fragmentary (Lin et al.2015). Autophagy has been shown to play a key role in both hepatocytes and nonparenchymal cells (i.e., macrophages and hepatic stem cells), affecting insulin sensitivity, lipid accumulation, hepatocyte injury, and the innate immune response.

Recent studies in mouse and cell models have shown that acute ethanol uptake activates autophagy in the liver (Ding et al 2010; Ni et al 2013; Lin et al 2013).

In contrast, chronic alcoholism has been shown to inhibit liver autophagy (Thomes et al 2015; Cho et al 2014). Inhibition of autophagy has been shown to exacerbate ethanol-driven steatosis and liver injury in mice (Ding et al.2010; Ni et al.2013; Lin et al.2013).

Conversely, pharmacological promotion of autophagy was shown to reduce ethanol-driven hepatic steatosis and liver injury (Lin et al.2013). As a result, autophagy is considered a protective mechanism against the cytotoxic effects of ethanol and has emerged as a target for the development of therapeutic agents for ALD.

As mentioned previously, the second leading cause of liver disease is obesity, which is widely spread in developed countries and is also increasing epidemiologically in developing countries, and causes so-called non-alcoholic fatty liver disease or NAFLD. Progression to steatohepatitis, cirrhosis and liver cancer is increasingly occurring worldwide. The biochemical and molecular mechanisms involved in this disorder overlap those generally described in ALD.

As alterations in amino acid metabolism are one hallmark of liver disease associated with both alcohol consumption and obesity development, characterized in particular by a reduced level of circulating branched-chain amino acids (BCAAs) (Charlton,2006), there is an increasing interest in developing new therapeutic approaches based on amino acid supplementation as a treatment for liver disease.

Disclosure of Invention

The object of the present specification is to provide a novel amino acid-based composition effective in the prevention and treatment of liver diseases.

In accordance with the present specification, the above objects are achieved thanks to the subject matter that is specifically mentioned in the appended claims, which are understood to constitute an integral part of the present disclosure.

One embodiment of the present specification provides a composition for treating a liver disease in a mammal comprising an active agent comprising the amino acids leucine, isoleucine, valine, threonine, lysine and the carboxylic acids citric acid, succinic acid, malic acid.

In one or more embodiments, the active agent of the composition further comprises one or more amino acids selected from the group consisting of histidine, phenylalanine, methionine, tryptophan, cysteine, and tyrosine.

In a preferred embodiment, the liver disease may be selected from alcoholic liver disease (ADL), non-alcoholic liver disease (NAFDL) lipodystrophy, hepatitis, cirrhosis, hepatocellular carcinoma (HCC).

Another embodiment of the present disclosure provides a method of treating a liver disease in a mammal, the method comprising: selecting a composition comprising an active agent comprising the amino acids leucine, isoleucine, valine, threonine, lysine and the carboxylic acids citric acid, succinic acid and malic acid; the composition is administered to treat a liver disease in a mammal.

Drawings

The invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

figure 1 shows gene expression shown as mRNA levels in HepG2 cells treated with different amino acid-based compositions and ethanol (EtOH) for 9 days (. <0.05 and × <0.01 vs CTRL cells).

FIG. 2 shows the levels of P62 protein in HepG2 cells treated with different amino acid-based compositions and EtOH for 9 days (. SP values <0.05 and. SP <0.01, relative to CTRL cells;. SP value <0.05, relative to EtOH-treated cells).

Detailed Description

In the following description, numerous specific details are set forth in order to provide a thorough understanding of some embodiments. The embodiments may be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase "in one embodiment" appearing in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

Alcoholic Liver Disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are major global health problems characterized by: pathological features develop over a long period of time, including hepatic steatosis, steatohepatitis, cirrhosis, and up to cancer.

Early events in the pathogenesis of ethanol, such as mitochondrial damage, Reactive Oxygen Species (ROS) generation, and fat accumulation, for example, are common features between ALD and NAFLD.

In addition, amino acid metabolism changes have been shown to be a distinguishing feature of liver disease, characterized by low levels of circulating Branched Chain Amino Acids (BCAAs), and BCAAs have been shown to correlate with a reduced frequency of cirrhosis complications when prescribed as maintenance therapy (Charlton 2006).

The inventors of the present application found that by adding a specific carboxylic acid to a composition comprising a combination of leucine, isoleucine, valine, threonine and lysine, a high effectiveness against liver diseases such as ALD and NAFLD, for example, can be achieved.

The compositions disclosed herein, which comprise as active agents the amino acids leucine, isoleucine, valine, threonine, lysine in combination with three carboxylic acids that are substrates of the tricarboxylic acid cycle, including specific amounts of citric acid, succinic acid and malic acid, have been shown to restore altered amino acid metabolism induced by ethanol, and thus prevent autophagy. The tricarboxylic acid cycle (TCA cycle), also known as the Krebs cycle (Krebs cycle) and the citric acid cycle, which is performed in the mitochondrial matrix is the second phase of cellular respiration, a three-phase process by which living cells break down organic fuel molecules in the presence of oxygen to harvest the energy required for their growth and division.

Compositions comprising the above-described active agents, as well as compositions comprising the above-described active agents containing other specific amino acids (listed in table 1 below), are significantly more effective than similar amino acid compositions that do not contain such specific carboxylic acids.

In one or more embodiments, in the compositions disclosed herein, the weight ratio between the total amount of citric acid, succinic acid and malic acid and the total amount of the amino acids leucine, isoleucine, valine, threonine, lysine is between 0.05 and 0.3, preferably between 0.1 and 0.25.

In one or more embodiments, the active agent may further comprise one or more amino acids selected from histidine, phenylalanine, methionine, tryptophan, cysteine, and tyrosine.

In one or more embodiments, the carboxylic acid included in the composition consists of citric acid, succinic acid, and malic acid.

In another embodiment, the active agent of the compositions disclosed herein may further comprise aspartic acid and/or ornithine L-alpha ketoglutaric acid (OKG).

According to one embodiment, the composition comprises an active agent consisting of: leucine, isoleucine, valine, threonine, lysine, histidine, phenylalanine, methionine, tryptophan, cysteine, and optionally tyrosine, as well as citric acid, succinic acid, and malic acid, which are the only amino acids included in the composition. Citric acid, succinic acid and malic acid may be the only carboxylic acids included in the composition.

In another embodiment, the composition may comprise the amino acids isoleucine, leucine and valine in an amount of 35% to 65% by weight, preferably 42% to 56% by weight, relative to the weight of the active agent.

In one or more embodiments, the weight ratio between leucine and citric acid is from 5 to 1, preferably from 2.50 to 3.50.

In another embodiment, the weight or molar amount of citric acid is greater than the weight or molar amount of each of malic acid and succinic acid. Preferably, the weight or molar amount of citric acid is higher than the total weight or molar amount of malic acid plus succinic acid. In another embodiment, the weight ratio between citric acid and the sum of malic acid and succinic acid is from 1.0 to 4.0, preferably from 1.5 to 2.5. In a preferred embodiment, the ratio of citric acid: malic acid: the weight ratio of succinic acid is 10:1:1 to 2:1.5:1.5, preferably 7:1:1 to 1.5:1:1, more preferably 5:1:1 to 3:1: 1. In a preferred embodiment, the ratio of citric acid: malic acid: the weight ratio of succinic acid is 4:1: 1.

According to some embodiments of the disclosure, the preferred isoleucine: leucine molar ratio of 0.2 to 0.7, preferably 0.30 to 0.60, and/or preferably valine: the leucine weight ratio is 0.2 to 0.70, preferably 0.30 to 0.65.

In another embodiment, the ratio of threonine: leucine molar ratio of 0.10 to 0.90, preferably 0.20 to 0.70, and/or lysine: the leucine weight ratio is 0.20 to 1.00, preferably 0.40 to 0.90.

In a preferred embodiment, the ratio between the total molar amount of citric acid, malic acid, succinic acid and the total molar amount of methionine, phenylalanine, histidine and tryptophan is higher than 1.35.

In one or more embodiments, the weight ratio between the sum of citric acid, malic acid, succinic acid and the sum of the branched amino acids leucine, isoleucine, valine is between 0.1 and 0.4, preferably between 0.15 and 0.35.

In another embodiment, the total weight amount of the branched chain amino acids leucine, isoleucine, valine plus threonine and lysine is higher than the total weight amount of the three carboxylic acids, e.g., citric acid, malic acid and succinic acid. Preferably, the weight amount of the single carboxylic acid (citric acid, succinic acid or malic acid) is less than the weight amount of each of the single amino acids leucine, isoleucine, valine, threonine and lysine.

In another embodiment, the total molar amount of lysine and threonine is higher than the total molar amount of the three carboxylic acids citric acid, succinic acid, malic acid. Preferably, the ratio between the total molar amount of the three carboxylic acids citric acid, succinic acid, malic acid and lysine and threonine is between 0.1 and 0.7, preferably between 0.15 and 0.55.

In one or more embodiments, the compositions disclosed herein further comprise a vitamin, preferably selected from vitamin B, for example being vitamin B₁And/or vitamin B₆。

In another embodiment of the present disclosure, the composition may comprise carbohydrates, additives and/or flavoring substances.

In a preferred embodiment, the composition is used exclusively for the prevention and/or treatment of liver diseases selected from the group consisting of: fatty liver disease, lipodystrophy, hepatitis, cirrhosis, hepatocellular carcinoma (HCC).

In one or more embodiments, the liver disease is fatty liver disease. Fatty liver disease can be caused by drinking (alcoholic fatty liver disease, ALD).

In one or more embodiments, the fatty liver disease is non-alcoholic fatty liver disease (NFLD).

In one or more embodiments, fatty liver disease can be caused by: drugs or toxins, such as, for example, amiodarone, methotrexate, diltiazem, antiretroviral therapy, glucocorticoids, tamoxifen, mycosis (mushroom poisoning).

Furthermore, the amino acid arginine is preferably avoided, in particular when preparing a composition and in particular an active agent according to the present disclosure. In addition, other amino acids that are preferably avoided by the compositions disclosed herein may be serine, proline, alanine. Such amino acids may be counterproductive or even harmful at certain concentrations or stoichiometric ratios in the composition.

The amino acids disclosed in the present application may be replaced by the corresponding pharmaceutically acceptable derivatives (i.e., salts).

As will be apparent hereinafter, administration of a composition according to the present disclosure is particularly effective in preventing and/or treating liver diseases.

In a preferred embodiment, the disclosed compositions are useful for treating Alcoholic Liver Disease (ALD).

According to another embodiment, the amino acid composition may comprise pharmaceutically acceptable excipients, such as, for example, proteins, vitamins, carbohydrates, natural and artificial sweeteners and/or flavoring substances. In a preferred embodiment, the pharmaceutically acceptable excipient may be selected from whey protein, maltodextrin, fructose, calcium caseinate, fish oil, sucralose, sucrose esters, vitamin D3, B vitamins.

For oral use, the composition according to the present description may be in the form of tablets, capsules, granules, gels, gelable powders or powders.

Further instructions regarding the amounts of the various amino acids provided by the compositions and the ratios therebetween are contained in the appended claims, which form an integral part of the technical teaching provided herein with respect to the present invention.

Examples

Table 1 shows two different amino acid based compositions tested in vitro on hepatocytes (HepG2 cells) and in vivo on ethanol-depleted rats, as disclosed below.

The composition, hereinafter referred to as "bcaaaem", comprises an active agent comprising the amino acids leucine, lysine, isoleucine, valine, threonine, cysteine, histidine, phenylalanine, methionine, tyrosine, tryptophan.

The composition referred to as "alpha 5m (α 5 m)" comprises an active agent containing the same amino acids plus citric, succinic and malic acids.

TABLE 1

The composition of table 1 above can be prepared by first screening all the components with a 0.8 mesh screen. To obtain a premix, each ingredient (in an amount of < 10% by weight of the total amount) was placed in a polyethylene bag together with a portion of L-lysine HCl to obtain 10% by weight of the total composition. The bag was then shaken manually for 5 minutes. The premix was then loaded in a mixer (Planetaria) together with the remaining ingredients and mixed for a period of 15 minutes at 120rpm to obtain a homogeneous final composition.

Method of producing a composite material

Animals and treatments

The experimental protocol was approved and implemented according to The European Community Council Directive (86/609/EEC) and Italian Ministry of health, 24.11.1986, and in compliance with The National Animal Protection Guidelines (The National Animal Protection Guidelines).

Male Wistar rats (3 months old) from Charles River (Calco, Como, Italy) were used for experimental analysis as disclosed below.

Animals were housed individually in clean polypropylene cages and divided into six groups:

1) the paired-fed group (paired-fed CTRL, n ═ 6) was fed with a control fluid diet in which ethanol (EtOH) was replaced by an isocaloric amount of maltodextran;

2) the EtOH group (EtOH, n ═ 7) was fed ad libitum with Lieber-DeCarli fluid diet containing EtOH [ increasing the amount of EtOH gradually, reaching 36% caloric intake after 1 week, corresponding to a final concentration of 6.2% (v/v) ];

3) a group of bcaaaem (bcaaaem, n ═ 6) fed on a control liquid diet, in which EtOH was replaced by isocaloric maltodextran, supplemented with a branched chain amino acid composition providing 1.5 g/kg/day BCAAem ("bcaaaem" in table 1);

4) an α 5 group (α 5m, n ═ 6) fed on a control liquid diet, in which EtOH was replaced by isocaloric maltodextran, supplemented with an amino acid composition providing 1.5 g/kg/day ("α 5 m" in table 1);

5) EtOH plus BCAAem group (EtOH + BCAAem, n ═ 7), which was fed ad libitum with Lieber-DeCarli fluid diet containing EtOH and BCAAem combination; and

6) EtOH plus α 5m group (EtOH + α 5m, n ═ 7), which was fed ad libitum with Lieber-DeCarli fluid diet containing EtOH and α 5m combination.

Sample preparation

Livers (n ═ 4 animals/group) were weighed, and treated in cold methanol: homogenized in water (v/v, 1:1) and extracted according to Want et al (Want et al.2013). The vacuum dried samples were suspended in methanol: 120 μ l/50mg tissue at 1mM TDFHA ═ 1:1, and centrifuged at 16,000g for 10 min at 4 ℃. Mu.l of the supernatant (surnatant) was directly loaded onto a UPLC mass spectrometer and analyzed as reported below. Four technical replicates were performed for each sample using three different methods.

Chromatography and quantification of amino acids in liver

Standard amino acids were purchased from Sigma (Milan, Italy). Each amino acid stock solution was prepared at 1mg/ml in water, diluted to a final concentration of 3 pmol/. mu.l, and injected directly into the TripleTOF 5600+ mass spectrometer (AB Sciex, Milan, Italy) via syringe at 10. mu.l/min. Therefore, the Declustering Potential (DP) and Collision Energy (CE) were optimized for each amino acid.

Next, three amino acid mixtures were prepared based on DP and CE values: MIX 1, comprising threonine, asparagine, tyrosine and serine, and substituted with DP: 30V, CE: 15V for analysis; MIX 2 comprising glycine, alanine, leucine, isoleucine, valine, proline, histidine, methionine, aspartic acid, glutamine and phenylalanine, and substituted with DP: 40V, CE: 15V for analysis; and MIX 3 comprising glutamic acid, lysine, arginine and tryptophan, and is substituted with DP: 80V, CE: analysis was carried out at 18V.

The source parameters are: gas 1: 33psi, gas 2: 58psi, curtain gas: 25psi, temperature: 500 ℃, and isff (ion spray Voltage Floating): 5500V.

To obtain calibration curves, different amounts (10, 33, 50, 100, 200, 400pm ol) of the three mixtures of technical quadruplicates were injected into the mass spectrometer after UPLC separation using UPLC 1290(Agilent Technologies Italia, Cernusco sul Naviglio, Milan, Italy). The column was from Waters, Acquity HSS T3 c 182.1x 100mm, 1.7 μm, with mobile phase a: 1mM TDFHA (tridecafluoroheptanoic acid) in water; b: 1mM TDFA in acetonitrile. A gradient of B from 12.5% to 26.5% in 4 minutes followed by a rise from 26.5% to 92% in 3.5 minutes was used to separate all amino acids with a flow rate of 0.35 ml/min and a column temperature of 65 ℃, as described (Le et al, 2014).

The autosampler setting was 4 ℃. Calibration curves were drawn by MultiQuant software version 2.1 (SCIEX) using chromatographic peak areas and weighted regression (all compounds 1/x except asparagine, tyrosine, valine and glutamic acid were fitted to 1/x 2). Quantitation (pmol) of each amino acid in rat liver samples was obtained by correlating the chromatographic peak area with the chromatographic peak area derived from an externally run calibration standard and normalizing for tissue (mg).

Cell culture and processing

Human HCC HepG2 cells were purchased from the American Type Culture Collection (HB-8065; ATCC, Manassas, Va.). Cells were routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (100U/mL) and streptomycin (100. mu.g/mL) at 37 ℃ in an atmosphere with 5% CO 2. Each 75cm²Flasks (Corning inc., Corning, NY) were seeded with 200 million HepG2 cells.

Six hours after inoculation, 75mM (0.34%) EtOH and 1% BCAAem or α 5m were added, alone or in combination. Untreated cells were plated as controls. Every 24 hours, the media in both the control and treatment flasks were replaced with fresh media with or without EtOH and bcaaaem or α 5m, respectively. Four days after inoculation, cells were trypsinized and inoculated into new flasks with 200 ten thousand viable cells per flask, and the medium was changed daily as described previously (Pochareddy et al 2012). Five days after the split procedure (9 days total, with or without EtOH, BCAAem or α 5m, or EtOH plus BCAAem or α 5m), cells were harvested for different assays as reported below.

Quantitative RT-PCR analysis

Quantitative RT-PCR reactions were performed as described (Tedesco et al.2008) and run on an iCycler iQ real-time PCR detection System (Bio-Rad) with an iQ SybreenI SuperMix (Bio-Rad; Segrate, Italy).

In short, use

Tissue mini kit (Qiagen, Milan, Italy) isolates RNA from tissue. cDNA was synthesized using the iScriptTM cDNA synthesis kit (Bio-Rad Laboratories, Segrate, Italy).

Primers were designed using Beacon Designer 2.6 software from Premier Biosoft International (sequences are reported in table 2 below). The cycle number at which multiple transcripts were detectable (threshold cycle, CT) was compared to the cycle number of TBP, referred to as Δ CT. The relative level of the gene was expressed as 2- (Δ Δ CT), where Δ Δ CT is equal to Δ CT of EtOH-or BCAAem-or CAA-mixture-treated rats (or treated HepG2 cells) minus Δ CT of control rats (or untreated HepG2 cells).

TABLE 2

T_aAnnealing temperature (. degree. C.); accession number PGC-1 α: NM-013261; accession number Tfam: NM-009360.4; accession number NRF1 NM-005011; accession number Cytc: JF 919224.1; accession number TBP: NG _051572 was used to normalize gene expression.

Western blot analysis

Protein extracts were obtained from liver with T-PER mammalian protein extraction reagent (Pierce, ThermoScientific, Rockford, USA) in the presence of protease and phosphatase inhibitor cocktail (Sigma Aldrich, Milan, Italy) as described by the manufacturer. Protein content was measured by a bicinchoninic acid protein assay (BCA, Pierce, Euroclone, Milan, Italy) and 50. mu.g of protein was run on SDS-PAGE under reducing conditions. The separated proteins were then electrophoretically transferred to nitrocellulose membranes (Bio-Rad Laboratories, Segrate, Italy). Specific antibodies for the target protein: anti-p 62 and anti-a-actin (all from Cell Signaling, Euroclone, Milan, Italy) were visualized, each antibody diluted 1: 1000. Immunostaining was detected for 1 hour at room temperature using horseradish peroxidase conjugated anti-rabbit or anti-mouse immunoglobulin. The amount of protein was measured using SuperSignal substrate (Pierce, Euroclone, Milan, Italy) and quantified by densitometry using IMAGEJ software image analyser.

Statistical analysis

For all gene expression data, values between control and treated cells were compared using a two-sided paired sample t-test. P values <0.05 were considered statistically significant.

Results

Composition α 5m more effectively restores liver mitochondrial biogenesis impaired by EtOH consumption than BCAAem compositions And function

The ability of the bcaaaem and α 5m compositions to improve mitochondrial biogenesis and function impaired due to EtOH exposure was evaluated. To investigate the molecular mechanisms involved in the action of bcaaaem and α 5m compositions, an in vitro model of liver EtOH toxicity was used.

To this end, liver HepG2 cells were exposed to 75mM EtOH with or without the bcaaaem α 5m composition for 9 days. Levels of proliferation-activating receptor gamma coactivator 1 alpha (PGC-1 alpha), nuclear respiration factor-1 (NRF-1), mitochondrial DNA transcription factor A (Tfam), and cyt c mRNA were unchanged or slightly lower in HepG2 cells exposed to 75mM EtOH for 9 days, compared to untreated control cells (FIG. 1).

However, administration of the BCAAem and α 5m composition increased PGC-1 α and Tfam mRNA levels for 9 days relative to untreated cells and EtOH-treated cells (FIG. 1).

Notably, the efficacy of the α 5m composition to improve liver mitochondrial biogenesis markers is statistically higher than that of BCAAem.

Autophagy assay

Autophagy flow can be inferred by combining several markers including p62/SQSTM1 protein levels and LC3II/LC3I ratios, Beclin1 and Atg7, and 4EBP1 phosphorylation (Klionsky et al, 2016). p62/SQSTM1 has been shown to be incorporated into autophagosomes and degraded in autolysosomes. Thus, decreased levels of p62/SQSTM1 protein indicated increased autophagy (Klionsky et al.2016).

As shown in figure 2 of the present application, p62 protein levels were lower in HepG2 cells exposed to 75mM EtOH for 9 days than in untreated control cells.

However, the p62 protein levels were higher after 9 days of administration of either bcaaaem or α 5m compositions relative to the p62 levels of untreated cells and EtOH treated cells.

Also in this case, the efficacy of the α 5m composition is statistically higher than that of the BCAAem composition.

Liver amino acid quantification

As disclosed above, altered amino acid metabolism, in particular low circulating BCAA levels, has been shown to be a distinctive feature of alcoholic liver disease (Charlton, 2006).

The data provided below refer to the effect of bcaaaem or α 5m compositions on amino acid metabolism in the liver of chronic EtOH-depleted rats.

For this purpose, the level of free amino acids in the liver tissue of rats depleted of EtOH alone or in combination with the test compositions was measured using chromatographic analysis.

As reported in table 3, supplementation with bcaaaem and α 5m compositions was not effective on arginine, leucine and tryptophan levels. In contrast, EtOH consumption results in reduced levels of these amino acids in the liver.

Interestingly, administration of bcaaaem and α 5m compositions in EtOH-depleted rats prevented arginine, leucine, and tryptophan reductions.

In addition, the liver of mice exposed to EtOH-containing diets had lower concentrations of isoleucine, serine, tyrosine and valine.

While BCAAem supplementation did not prevent the decline, α 5m supplementation was instead also effective in preventing EtOH-induced isoleucine and valine reductions.

The concentrations of the remaining amino acids did not differ statistically between groups.

These results are consistent with the specific ability of the α 5m composition to renormalize BCAA levels in liver samples of EtOH-depleted rats compared to the BCAAem composition.

TABLE 3

Values are reported as mean ± SD (pmol/mg tissue), n-4 animals/group; relative to CTRL groups, P<0.05；^#P value relative to EtOH group<0.05。

In summary, in vitro and in vivo results indicate that dietary supplementation with compositions comprising active agents comprising a combination of leucine, isoleucine, valine, threonine, lysine, citric acid, succinic acid and malic acid is significantly effective in preventing mitochondrial damage in hepatocytes exposed to EtOH.

This effect was also accompanied by a decrease in autophagy (i.e., an increase in p62 protein levels) that was increased by EtOH exposure (i.e., a decrease in p62 protein levels).

EtOH itself can enhance autophagy to compensate for EtOH toxicity and in particular mitochondrial damage evident in alcohol-exposed hepatocytes and in the livers of alcohol-consuming animals.

Indeed, autophagy is a cellular mechanism aimed at eliminating dysfunctional organelles (including mitochondria). When hepatocytes are exposed to alcohol and mitochondrial function is reduced, increased autophagy, as also disclosed herein, can be used to eliminate dysfunctional mitochondria.

The amino acid composition disclosed herein, which comprises leucine, isoleucine, valine, threonine, lysine in combination with citric acid, succinic acid and malic acid, is capable of preventing mitochondrial damage such that cells do not require autophagy to maintain their viable function. Thus, autophagy is reduced (as shown by the increased levels of p62 protein shown in the present application).

Furthermore, and most importantly, the disclosed compositions have been found to be very effective in restoring free BCAA, arginine, and tryptophan concentrations in the livers of EtOH-depleted rats.

Notably, preliminary results indicate that the amino acid composition disclosed herein comprising leucine, isoleucine, valine, threonine, lysine in combination with citric, succinic, and malic acid is also able to reduce the diameter of lipid droplets in hepatocytes of mice exposed to high-fat diet (HFD, 60% calories from fat) for 6 months, which are a widely used NAFLD mouse model. Taken together, these results strongly support the following notions: the amino acid compositions disclosed herein and having activity in alcohol-dependent hepatotoxicity may be healthy in preventing the onset and exacerbation of NAFLD.

As is apparent from the foregoing, the composition according to the present disclosure may be useful for preventing and/or treating liver diseases that affect a large number of adult populations on a global scale and increase social costs.

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Sequence listing

<110> Professional Dietetics

<120> composition for treating liver diseases

<130> BWO21147

<160> 10

<170> BiSSAP 1.3.6

<210> 1

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> PGC1 alpha sense 5'-3'

<400> 1

gaccccagag tcaccaaatg ac 22

<210> 2

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> antisense 5' -3' of PGC1 alpha '

<400> 2

ttggttggct ttatgaggag ga 22

<210> 3

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Tfam sense 5'-3'

<400> 3

agattggggt cgggtcac 18

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Tfam antisense 5'-3'

<400> 4

gacaacttgc caagacagat g 21

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> NRF1 sense 5'-3'

<400> 5

actcgtgtgg gacagcaagc 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> NRF1 antisense 5'-3'

<400> 6

atggtgagag gcggcagttc 20

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Cytc sense 5'-3'

<400> 7

cgttgtgcca gcgactaaaa a 21

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Cytc antisense 3'-5'

<400> 8

ttccgcccaa agagacca 18

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> TBP sense 5'-3'

<400> 9

aggcaccaca gctcttccac 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> TBP antisense 5'-3'

<400> 10

cccagaactc tccgaagctg 20

Claims

1. A composition for use in the prevention and/or treatment of liver disease in a mammal, the composition comprising an active agent comprising the amino acids leucine, isoleucine, valine, threonine, lysine and the carboxylic acids citric acid, succinic acid, malic acid.

2. Composition for use according to claim 1, wherein the weight ratio between the total amount of citric acid, malic acid, succinic acid and the total amount of leucine, isoleucine, valine, lysine, threonine is between 0.05 and 0.3, preferably between 0.1 and 0.25.

3. Composition for use according to any one of the preceding claims, wherein the weight ratio between the total amount of citric acid, malic acid, succinic acid and the total amount of leucine, isoleucine, valine is between 0.1 and 0.4, preferably between 0.15 and 0.35.

4. Composition for use according to any one of the preceding claims, in which the weight ratio between citric acid and the sum of malic acid and succinic acid is from 1.0 to 4.0, preferably from 1.5 to 2.5.

5. The composition for use according to any one of the preceding claims, wherein the ratio of citric acid: malic acid: the weight ratio of succinic acid is 10:1:1 to 2:1.5:1.5, preferably 7:1:1 to 1.5:1:1, more preferably 5:1:1 to 3:1: 1.

6. The composition for use according to any one of the preceding claims, wherein the active agent further comprises at least one amino acid selected from histidine, phenylalanine, methionine, tryptophan, tyrosine, cysteine.

7. The composition for use according to any one of the preceding claims, wherein the active agent further comprises histidine, phenylalanine, methionine, tryptophan, cysteine and optionally tyrosine.

8. Composition for use according to any one of the preceding claims, wherein the ratio between the total molar amount of citric acid, malic acid, succinic acid and the total molar amount of methionine, phenylalanine, histidine and tryptophan is higher than 1.35.

9. Composition for use according to any one of the preceding claims, in which the ratio between the total molar amount of the three carboxylic acids citric acid, succinic acid, malic acid and lysine and threonine is between 0.10 and 0.70, preferably between 0.15 and 0.55.

10. Composition for use according to any one of the preceding claims, wherein the weight or molar amount of citric acid is higher than the total weight or total molar amount of both malic acid and succinic acid.

11. Composition for use according to any one of the preceding claims, in which the weight ratio between leucine and citric acid is from 5 to 1, preferably from 2.50 to 3.50.

12. Composition for use according to any one of the preceding claims, wherein the composition further comprises one or more vitamins, preferably selected from vitamin B, more preferably vitamin B1 and/or vitamin B6.

13. The composition for use according to any one of claims 1 to 12, wherein the liver disease is selected from fatty liver disease, lipodystrophy, hepatitis, cirrhosis, hepatocellular carcinoma (HCC).

14. The composition for use according to claim 13, wherein the liver disease is selected from the group consisting of Alcoholic Liver Disease (ALD) and non-alcoholic fatty liver disease (NAFLD).